How does the facial skeleton acquire its characteristic shape? What is the developmental basis of craniofacial birth defects in humans? The long-term goal of this proposal is to understand how epithelia communicate with skeletal precursors to generate precise cellular arrangements of cartilage and bone in the face. Zebrafish is an excellent system to model vertebrate development. This proposal uses strengths of the zebrafish system - powerful forward genetics, the ability to manipulate embryos, and in vivo imaging - to understand the tissue interactions, molecular signalling, and cell biology that specify cartilage shape. Development of the facial skeleton involves an interplay between intrinsic factors that give skeletal precursors a set identity and extrinsic signals from neighboring epithelia. The first aim is to examine how skeletal precursors acquire dorsal identity, and then how this dorsal identity allows them to respond to specific signals from neighboring epithelia. In a newly identified pucker mutant, the dorsal skeleton is transformed to a ventral character, and the expression of ventral dlx genes is expanded dorsally. The cloning and characterization of pucker will provide insights into how the dorsal skeleton is specified and shaped. In the second aim, the identity of the endodermal signals that act on skeletal precursors is investigated. Avian experiments demonstrate a role for endoderm in skeletal patterning, yet it is unclear whether the endoderm signals directly to skeletal precursors at pharyngeal arch stages. Ablation and graft experiments will test that the endoderm directly patterns facial cartilage at arch stages, and gain-of-function studies will test that the endoderm patterns cartilage by secreting distinct combinations of Fgfs and Hhs. In the third aim, the cell biology of epithelial-mesenchymal interactions in the face is investigated. Time-lapse imaging will test the model that endodermal epithelia initiate morphology and polarity changes in preskeletal mesenchyme that lead to the formation of cell condensations, a poorly understood developmental intermediate. The role of the endoderm in later cell rearrangements that refine cartilage shape will also be examined. The completion of these aims will further our understanding of facial skeleton development and lead to the better diagnosis, prevention, and treatment of human craniofacial disorders. In addition, the basic developmental knowledge obtained will be essential for the design of cell-based therapies aimed at regenerating the facial skeleton.